The prognosis of patients receiving chemotherapy for advanced non-small cell lung cancer (NSCLC) remains very poor. Emerging evidence indicates that the response rate of NSCLC patients to currently used anticancer drugs could be increased by preselecting the most responsive patients for personalized therapy. This requires molecular biomarkers indicative of sensitivity to the specific drug. We have recently identified that high ISG15 expression indicates sensitivity of lung cancer cells to topotecan (TPT). Intravenous (IV) infusion of TPT is FDA approved for lung cancer but its use is limited due to a severe hematological toxicity in some patients. This application aims to improve the efficacy and toxicity of TPT through three innovative approaches: 1) A bioengineering concept driven strategy to manufacture and deliver a spray-dried powder of TPT via inhalation, which allows effective drug concentration in the lungs while reducing it in the systemic circulation. 2) A novel biomarker for preselecting the most TPT-sensitive NSCLC cells. 3) The use of a unique orthotropic lung cancer model in which human NSCLC cells are engrafted throughout the lungs of the nude rat, a semi- natural system that captures the impact of the tumor-microenvironment on tumor growth and treatment response. Studies under Aim-1 will produce a spray-dried powder formulation of TPT, evaluate its aerosol property, and characterize its plasma and pulmonary pharmacokinetics with respect to the standard IV TPT delivery in the nude rat. Under Aim-2 human NSCLC cell lines that are preselected for TPT-sensitivity (n=2) or resistance (n=2) based on ISG15 expression will be instilled into the lungs of nude rats via tracheal intubation. After 3 weeks of tumor growth, the rats will be treated once-a-week for 4 weeks with vehicle (filtered air), 3 different doses of inhaled-TPT, or an IV-TPT, and the toxicity and efficacy to suppress tumor growth will be determined. Aim-3 will use nude rats engrafted with the 2 most TPT-sensitive cell lines to compare the impact of vehicle, inhaled-TPT, and IV TPT therapy in improving lung cancer survival. Additional studies under this aim will use subcutaneous xenografts of the two TPT-sensitive cell lines to evaluate the impact of these treatments in suppressing extrapulmonary growth of lung cancer. Finally, studies under Aim-4 will determine TPT-sensitivity of 36 NSCLC cell lines in vitro, and screen the genome-wide gene and microRNA expression of TPT-sensitive and resistant cell lines (n=10 each). The goal is to identify additional biomarkers that could discriminate NSCLC cells by TPT-sensitivity. The potential of the top biomarkers to complement and improve the ISG15-based preselection of lung cancer patients that are most responsive to TPT therapy will be determined using lung tumor-normal pairs from 100 NSCLC patient. These studies have the potential to significantly improve the efficacy and utility of this potent anticancer drug and due to its approval and availability in the clinic could immediately impact NSCLC therapy.